Compositions of digestive enzymes and salts of bile acids and process for preparation thereof

ABSTRACT

Disclosed are gastric acid-resistant polymer-coated buffered digestive enzymes/ursodeoxycholate compositions, process for their preparations and methods of treating digestive disorders, pancreatic enzyme insufficiency, impaired liver function, cystic fibrosis, for regulating the absorption of dietary iron and cholesterol, and for dissolving gallstones by administering the compositions to a mammal in need of such treatment.

This Application is a continuation-in-part of application Ser. No.8/104,655, filed Aug. 11, 1993, now U.S. Pat. No. 5,324,514,which inturn is a divisional of application Ser. No. 07/901,734, filed Jun. 22,1992 now U.S. Pat. No. 5,260,074 issued as U.S. Pat. No. 5,260,074.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to buffered digestive enzymes and salts of bileacids, and more particularly salts of ursodeoxycholic acid compositionsfor ingestion by a mammal, a process for preparing said compositions,and a method for treating digestive disorders, pancreatic enzymeinsufficiency, impaired liver function, pancreatitis, cystic fibrosis,regulating dietary cholesterol absorption and for dissolving gallstonesby administering said compositions to a mammal in need of suchtreatment.

2. Reported Developments

It is known in the prior art that pancreatic enzymes administered tomammals can remedy enzyme deficiency caused by various diseasedconditions of the pancreas, such as cystic fibrosis, pancreatitis,pancreatic enzyme deficiency and old age. Oral administration ofcompositions containing these enzymes requires the presence of certainconditions in order for them to be safe and effective as will bedescribed hereunder.

Pancreatic enzymes produced by the patient's pancreas are released intothe duodenum, the pH of which is close to neutral or slightly alkaline.Under these pH conditions the enzymes are active and digestion of thefood by the enzymes proceeds normally in the upper segment of theintestine. However, when pancreatic enzymes are administered exogenouslyto the patient, the gastric conditions in the stomach, namely thepresence of acid and pepsin, will irreversibly inactive the enzymes.Therefore, orally administered enzymes must be protected against gastricinactivation so that they remain intact during their transit through thestomach into the duodenum.

Once the exogenously introduced enzymes reach the duodenum, anotherrequirement must be satisfied: the enzymes must be released from theirprotective environment and intimately mixed with the food transferredfrom the stomach to effect digestion at a neutral to slightly alkalinecondition.

U.S. Pat. No. 4,079,125 incorporated herein by reference, addressesthese requirements in a composition containing these enzymes andprovides preparative methods for making the compositions. Thecompositions provided by said patent comprise: an enzyme concentrate ina binder selected from the group consisting of polyvinylpyrrolidone,microcrystalline cellulose, cellulose acetate phthalate, methylcelluloseand alginic acid; a stabilizer selected from the group consisting ofcalcium carbonate, polyvinylpyrrolidone, cellulose acetate phthalate,methylcellulose, starch and modified starches and alginic acid; adisintegrant selected from the group consisting of citric acid, sodiumcarbonate, sodium bicarbonate, calcium carbonate, starch and modifiedstarches and alginic acid; said mixture is coated with a non-porous,pharmaceutically acceptable enteric coating polymer which is insolublein the pH range of from about 1.5 to about 5 normally present in gastricfluids, and soluble at a pH of from about 6 to about 9, the normal pHrange for mammalian intestinal fluids.

The orally administered composition passes through the stomach whilebeing protected against the acidic environment by its acid-insolublecoating which then disintegrates in the neutral to basic environment ofthe upper intestine releasing the enzymes from the composition. Theprocess of making the compositions includes the provision of using asolvent and avoiding the presence of water in the blending step of theenzyme/binder/disintegrant, since it is believed that water deactivatessome of the enzymes.

Contrary to the teaching of U.S. Pat. No. 4,079,125, it has now beendiscovered that the complete exclusion of the water (anhydrouscondition) during the process of preparing the buffered enzymes/salts ofursodeoxycholic acid compositions in the form of microtablets andmicrospheres, leads to products that are extremely friable, tend tocrumble into pieces upon drying in a fluidized bed dryer or conventionalcoating pan and disintegrate upon initiation of the polymer coatingstep. This results in large amounts of dust and agglomeration of thebeads into multiplets during the process as well as improper doses ofthe enzymes upon administration to the patient when quality controlfails adequately to sort out and discard rejects.

It is also known that ursodeoxycholic acid (hereinafter UDCA or bileacid) is capable of augmenting liver function, dissolving gallstones andimproving the nutritional state of patients having hepatobiliarycomplications associated with cystic fibrosis. See for example,Ursodeoxycholic Acid Dissolution of Gallstones in Cystic Fibrosis, Sahl,B., Howat, J., Webb, K., Thorax, 43:490-1 (1988); Effects ofUrsodeoxycholic Acid Therapy for Liver Disease Associated with CysticFibrosis, Colombo, C., Setcheil, K. D., Podda, M., Crosignani, A., RodaA., Curcio, L., Ronchi, M. and Giunta, A., The Journal of Pediatrics,117:482-489 (1990); Effects of Ursodeoxycholic Acid Treatment onNutrition and Liver Function in Patients with Cystic Fibrosis andLongstanding Cholestasis. Cotting, J., Lentze, M. J. and Reichen, J.,Gut 31:918-921 (1990). Also, UDCA has recently gained acceptance as aneffective therapeutic modality to dissolve small to medium sizecholesterol gallstones in gallstone afflicted patients. See for example,The Effect of High and Low Doses of Ursodeoxycholic Acid on GallstoneDissolution in Humans, Salen, G., Colalillo, A., Verga, D., Bagan, E.,Tint, G. S. and Shefer, S., Gastro,, 78:1412-1418 ( 1980);Ursodeoxycholic Acid: A Clinical Trial of a Safe and Effective Agent forDissolving Cholesterol Gallstones, Tint, G. S., Salen, G., Colalillo,A., Graber, D., Verga, D. Speck, J. and Shefer, S., Annals of InternalMedicine, 91:1007-1018 (1986); Clinical Perspective on the Treatment ofGallstones with Ursodeoxycholic Acid, Salen, G., J. Clin.Gastroenterology, 10 (Suppl. 2):S12-17 (1988); Nonsurgical Treatment ofGallstones, Salen, G. and Tint, G. S., New England J. Med, 320:665-66(1989); and Reducing Cholesterol Levels, Weigand, A. H., U.S. Pat. No.3,859,437. The recommended dosage is 10 to 15 mg/kg of body weight. Insome patients much higher dosages (for example, about 30 mg/kg of bodyweight) are required to achieve limited benefits. However, in somepatients undesirable side effects (such as, severe diarrhea) seriouslylimit the use of this drug. The reasons for this wide variation ofdosage requirements for therapeutic effectiveness and associated sideeffects are not completely understood. One hypothesis is that the freeacidic form of UDCA is only partially neutralized in the upper intestineto its sodium salt form due to deficiencies in bicarbonate in some ofthe patients. The residual free acidic (insoluble) form of UDCA ispoorly absorbed from the intestine, and a good portion of theadministered dosage is excreted intact with feces. When a higher dosageof the acidic form of UDCA is administered to the patient, a largeportion of it is neutralized in the distal parts of the intestine whichin turn induces diarrhea, a highly undesirable side effect. Also, if theacidic form of UDCA is to be converted into its salt form in theduodenum, it will temporarily exhaust the buffering capacity of theduodenum and it will render the upper intestine partially acidic. Theacidic pH impedes the function of the pancreatic enzymes and UDCA cannotemulsify fats and facilitate the hydrolysis of lipids. Furthermore, themany therapeutic benefits derived from the salt forms of UDCA cannot berealized. It should then follow, accordingly, that the salt forms ofUDCA should be administered to patients in need of UDCA. U.S. Pat. No.3,859,437 recommends the administration of a "small but effectiveamount, sufficient to effect a reduction in the cholesterol level ofsaid human being of the compound 3α 7β-dihydroxy-5β-cholanic acid (UDCA)and the non-toxic pharmaceutically acceptable salts thereof". However,administering the salt form of UDCA to patients has no advantage overthe acidic form of UDCA and does not accomplish the desired resultssince the salt form of UDCA is converted back to the insoluble acidicform of UDCA by gastric acidity. Furthermore, the salt forms, i.e.,sodium or potassium, of UDCA are extremely bitter-tasting, and in mostpatients cause esophageal reflux, nausea and vomiting. Because of thesehighly undesirable organoleptic and gastric side effects, the salt formsof UDCA has not gained therapeutic utility in the treatment of biliarydiseases.

Pancreatic enzymes and salts of UDCA complement one another in thedigestive system of a mammal. A dietary supplement containing both theenzymes and salts of UDCA would provide in a convenient predetermineddose the remedy needed to treat the above-described diseased states.However, the acidic form of UDCA is incompatible with pancreaticenzymes. Pancreatic enzymes/UDCA compositions have a pH of about 5 to5.5. Under these acidic conditions most pancreatic enzymes show a lowbiological activity of about 10% to 40%. Lipase is especially affectedby the low pH for the reasons that: UDCA is only sparingly soluble inaqueous media and is inefficient to emulsify fats; and the acidic UDCAinactivates lipase since lipase requires a basic pH for biologicalactivity.

Pancreatic enzymes/UDCA containing compositions also lack sufficientshelf-life due to the denaturing and detergent effects of UDCA on thepancreatic enzymes. Because of these incompatibilities between UDCA andpancreatic enzymes the many benefits derivable from their combinationscould not be realized by the prior art.

It has now been discovered that the problems associated individuallywith enteric coated microtablets and microspheres containing pancreaticenzymes and compositions containing UDCA, may be overcome in a dietarysupplement containing both the pancreatic enzymes and a salt of UDCA. Inaccordance with the discovery, UDCA is first converted to apharmaceutically acceptable salt, such as the sodium or potassium saltand then used in a combination with pancreatic enzymes in a composition.Such salts are highly effective to emulsify fats and lipids at a basicpH and facilitate the hydrolysis of the emulsified fat globules. As aresult, fat digestion is greatly enhanced. The salts are also moreeffective than the insoluble acidic form of UDCA to lyse mucus whichblocks the intestinal surfaces and prevents absorption of metabolitesthat results in poor nutrition in cystic fibrosis children.

Pancreatic enzymes then are combined with a salt of UDCA and bufferedwith a biologically compatible, pharmaceutically acceptable buffer thatprevents deactivation of the enzymes and preserves the naturalbiological activities of both the buffered enzymes and the salt of UDCA.The pancreatic enzymes/salt of UDCA composition can be prepared intomicrotablets and microspheres in the presence of moisture withoutinactivation of the enzymes/bile salt composition thereby resulting inproducts that do not crumble upon drying or disintegrate upon initiationof the polymer coating procedure. The bitter taste and associatedgastric disadvantages of UDCA salts are also eliminated by the polymercoating which prevents solubilization of the product in the mouth andstomach of the patient.

Still further, it has been discovered that microspheres in the range of10 to 80 mesh size (about 2.0 to 0.177 mm range) can be preparedutilizing bile salts as seeds to build up the microspheres. Such smallparticle size microspheres are especially beneficial for use to treatpancreatic enzymes/bile salt deficiencies in cystic fibrosis children.

SUMMARY OF THE INVENTION

The invention will be described with particular reference to salts ofursodeoxycholic acid, however, it is to be understood that salts ofother bile acids such as cholic acid, deoxycholic acid, chenodeoxycholicacid and their glycyl, taurine, methylglycyl and methyltaurineconjugates and salt complexes thereof including their isomers may beused as well.

In accordance with the present invention, buffered digestiveenzymes/salt of UDCA compositions are provided which possess desirablecharacteristics heretofore absent in proposed or existing prior artproducts.

The buffered digestive enzymes/salt of UDCA is instantly soluble inwater, while UDCA alone or in combination with a digestive enzyme isessentially insoluble.

Only the ionized or salt form of UDCA or the conjugated derivatives ofUDCA are absorbed from the intestine, while the acidic form of UDCA isinsoluble and passes through the intestine intact, unless it isconverted to the sodium salt by the intestinal buffers. However, manypatients, such as patients with cystic fibrosis, pancreatitis, BillrothI & II diseases and some elderly people, are partially deficient inbicarbonate secretion and lack neutralization capacity to convert theacidic form of UDCA to the sodium salt of UDCA. These patients will onlypartially benefit from UDCA therapy. The salt of UDCA-containingcomposition of the present invention overcomes this problem by beinginstantly soluble in the intestinal juices and absorbable from theintestine. Additionally, the composition also provides extra bufferingcapacity to neutralize the enteric coating polymer and the acid chymethat is present in the intestine and greatly facilitates the efficientdigestion of fats and lipids in the upper intestine.

The buffered digestive enzymes/salt of UDCA composition ismicroencapsulated and coated with an acid-resistant polymer-coating,which protects the composition from gastric acid and from conversion ofthe salt of UDCA to the acidic farm of UDCA. The polymer-coatedmicrocapsules are tasteless and the problem associated with theoffensive bitter taste of the uncoated acidic form or the uncoated saltof UDCA is thereby alleviated.

The microcapsules uniformly disperse with the food in the stomach anddeliver high levels of biologically active buffered digestiveenzymes/salts of UDCA into the duodenum. Once in the duodenum, thepolymer coating dissolves within about 10 to 30 minutes and the bufferedenzymes/salts of UDCA are released to enhance digestion of fats andlipids. As a result, the natural digestive conditions in the intestineare re-established. Epigastric pain, cramps, bloating, flatulence andstool frequency associated with maldigestion of fatty foods are reduced.

Soluble salts of UDCA and conjugated derivatives of UDCA are absorbedmore efficiently and in a greater quantity from the intestine than theinsoluble acidic form of UDCA, resulting in a more efficient stimulationof the liver enzymes to conjugate ursodiol (UDCA). The increasedconcentration of the conjugated ursodiol stimulates bile flow, enhancesthe displacement of toxic bile acid metabolites from the hepatocytes,decreases cholesterol secretion into bile, alters thecholesterol/phospholipid ratio of secreted bile and decreases theabsorption of dietary cholesterol from the intestine. The overall resultis decreased biliary cholesterol saturation, increased bile flow,dissolution of already formed cholesterol gallstones and protection ofthe liver from accumulated toxic metabolites.

Most recently, it has also been discovered that the enteric coatingpolymers employed to protect the pancreatic enzymes against gastricinactivation during gastric transit, are also acidic in nature, andrequire substantial quantity of base to be neutralized. For example,some of the commercially marketed products, such as CREON®, COTAZYM-S®and PANCREASE® when ground up and dissolved in water had pH's of 5.5,5.4 and 5.6, respectively. At this acidic pH's, lipase, an essentialenzyme that is required by cystic fibrosis patients, is rendered totallyineffective. Therefore, in order to compensate for the acidic nature ofthe enteric coated polymer, one needs to include extra bufferingcapacity in the composition to neutralize the acidic enteric coat andprovide a basic environment for lipase to exert its enzymatic activity.

Furthermore, recent findings indicate that buffer deficiency is moreserious in certain patients than believed heretofore. For example,cystic fibrosis patients are deficient in bicarbonate secretion andtheir upper intestinal pH's are in the range of 4.5 to 6.5; some of thealcohol induced cholestatic liver diseased patients duodenal pH's arealso in the less than neutral range, such as pH 6.0 to 7.6. Even whenthe pancreas and the gallbladder are maximally stimulated with secretionand cholecystokinin hormones, the intestinal pH's seldom exceed pH 7.0in cystic fibrosis and 7.6 in alcoholic liver diseased patients.

Because it is difficult to predict the extent of bicarbonate deficiencyin these patients population without intubation and collection ofintestinal juices for pH and bicarbonate assays, it is necessary toassure that adequate amount of buffer is administered with the exogenouspancreatic enzymes and bile salts compositions.

Accordingly, the composition of the present invention incorporates 5 to40% w/w of a buffering agent, based on the total weight of thecomposition, to provide optimal conditions for maximal biologicalactivity of the exogenously administered pancreatic enzymes and bilesalts.

The buffered digestive enzymes/salt of UDCA composition for thetreatment of enzyme/UDCA deficient mammals comprises a blend ofingredients and a coating therefor expressed in weight per weightpercentages based on the total weight of the composition:

a) from about 10 to about 90.0% of a concentrate of an enzyme selectedfrom the group consisting of pancreatin, pancreatic proteases, lipases,nucleases and amylases;

b) from about 0 to about 75%, and preferably of from about 0.3 to about75% of a UDCA salt in powder form;

c) from about 5 to about 40% of a buffering agent selected from thegroup consisting of sodium carbonate (anhydrous powder), sodiumbicarbonate, potassium carbonate, ammonium carbonate, tromethamine,tris-carbonate (Di[tris(hydroxymethyl)aminomethane] carbonate),tris-glycine buffer (0.25 mole tris-base and 1.92 mole of glycine, pH8.3), di-, tri-, and poly-arginine in the molecular range of 350 to50,000 daltons, di-, tri-, and poly-lysine in the molecular range of 290to 15,000 daltons, diethylamine and triethanolamine;

d) from about 0 to about 16%, and preferably from about 0.9 to about 16%of a disintegrant selected from the group consisting of starch andmodified starches, microcrystalline cellulose, and propylene glycolalginate;

e) from about 0.3 to about 15% of an adhesive polymer selected from thegroup consisting of polyvinylpyrrolidone; microcrystalline cellulose;hydroxypropyl cellulose; cellulose acetate phthalate: and a 60:40 blendof methyl cellulose/hydroxypropyl methyl cellulose; and

f) from about 7.0 to about 15% of a non-porous, pharmaceuticallyacceptable gastric acid-resistant polymer-coating which contains lessthan 2% talc and which is insoluble in the pH range of from about 1.5 toabout 5 but is soluble in the pH range of from about 5.5 to about 9.

The digestive enzymes of the present invention include Pancreatin ofmultiple strength, pancrelipase, trypsin, chymotrypsin, chymotrypsin B,pancreatopeptidase, carboxypeptidase A, carboxypeptidase B, glycerolester hydrolase, phospholipase A2, sterol ester hydrolase, ribonuclease,deoxyribonuclease, α-amylase, papain, chymopapain, bromelain, ficin,β-amylase, cellulase, and βgalactosidase (lactase).

The salts of UDCA includes sodium, potassium, ferrous, ammonium,tromethamine, ethanolamine, diethanolamine and triethanolamine salts orsalt complexes of UDCA.

In accordance with the present invention, the buffered enzyme/bile saltcomposition is prepared by a process comprising the steps of:

a) blending dry, powdery ingredients selected from the group consistingof (i) from about 10 to about 90% w/w of an enzyme from the groupconsisting of pancreatic proteases, lipases, nucleases and amylases;(ii) from about 0 to about 75% w/w, and preferably of from about 0.3 toabout 75% of a salt or salt complexes of UDCA selected from the groupconsisting of sodium, potassium, ferrous, ammonium, tromethamine,ethanolamine, diethanolamine and triethanolamine; (iii) from about 5 toabout 40% of a buffering agent selected from the group consisting ofsodium carbonate (anhydrous powder), sodium bicarbonate, potassiumcarbonate, ammonium carbonate, tromethamine, tris-carbonate(Di[tris(hydroxymethyl)-aminomethane]carbonate), tris-glycine buffer(0.25 mole tris-base and 1.92 mole of glycine, pH 8.3), di-, tri-, andpoly-arginine in the molecular range of 350 to 50,000 daltons, di-,tri-, and poly-lysine in the moleculare range of 290 to 15,000 daltons,diethylamine and triethanolamine; (iv) of from about 0 to about 16% w/w,and preferably from about 0.9 to about 16% of a disintegrant selectedfrom the group consisting of starch, modified starches, microcrystallinecellulose and propylene glycol alginate; and (v) from about 0.3% toabout 15% w/w of an adhesive polymer selected from the group consistingof polyvinylpyrrolidone, cellulose acetate phthalate, hydroxypropylcellulose and methylcellulose;

b) wetting said blended ingredients with a liquid to cause the blend tostick together, wherein said liquid is selected from the groupconsisting of: 1%-25% w/w ethanol/75%-99% w/w 2-propanol/0.05%-1.5% w/wwater; 98%`99% w/w 2-propanol/0.05%-1.5% w/w water; and 1%- 25% w/wmethanol/0.05%-1.5% w/w water/75%-98% w/w 2 propanol/1%-5% w/wethylacetate;

c) extruding the liquid-wetted blend through a 10 or a 18 mesh S/Sscreen;

d) converting the extruded segments to a uniform diameter particle size;

e) compacting the uniform particles to spherical particles;

f) drying the spherical particles;

g) separating the spherical particles if not of uniform size accordingto desired sizes using U.S. Standard sieve screens;

h) coating the particles with a gastric acid-resistant polymer thatdissolves under neutral or slightly basic conditions; and

i) drying the polymer-coated spherical particles.

DETAILED DESCRIPTION OF THE INVENTION

In preparing the buffered pancreatic enzymes/bile salt containingmicrospheres of the present invention utilizing the extrusion, uni-sizerand marumerization process (later described) moisture must be includedin the liquid or solvent-adhesive composition to render the adhesivepolymer sticky enough to bind the buffered enzymes/bile salt-containingfluffy powder into a pliable, solid mass. This prevents the crumbling ofthe microspheres during the drying and coating steps as well as allowsthe preparation of much smaller particle size microspheres, i.e. in therange of 10 to 80 mesh. Accordingly, it was found that the moisturelevel during the preparation of the composition should be in the rangeof from about 0.05% w/w to about 1.5% w/w, preferably, in the range of0.2%w/w to 1.5% w/w, and most preferably in the range of 0.2% w/w to1.0% w/w. When the compositions contained such amounts of moisture, themicrospheres were found to be stable on aging and biological activitywas preserved as long as the moisture level did not exceed about 1.5%w/w of the total composition.

Further reference is now made to the process of preparing compositionsof the present invention.

The process for the manufacture of microspheres consists of:

1) Blending the dry, powdery ingredients together in a conventionalblender and wetting the composition with a suitable liquid composition,hereinbefore described, that causes the dry blend to stick together. Thestickiness of the blend can be tested by compressing a handful of theblend in the palm of the hand. If the composition is compressible andsticks together but readily crumbles when squeezed between the fingers,sufficient liquid has been added to the composition for processing inthe subsequent granulation step.

2) Extruding the liquid moistened composition through a 10 or a 18 meshS/S screen using an oscillating/reciprocating granulator or a twin-screwextruder at a medium-to-high speed.

3) Classifying the extruded particles in a so-called "uni-sizer vessel"that rotates at 15 to 45 rpm for about 5 to 10 minutes. (The particlesin the "uni-sizer vessel" are converted to a uniform diameter particlesize.)

4) Compacting the uniform particles in a marumerizer, (a cylindricalvessel with a rotating disk at the bottom) for about 15 to 70 seconds.An alternative method of compacting the microspheres can also beachieved in a rotating conventional coating pan. In this case, theparticles are tumbled in the pan for about 15 to 30 minutes,occasionally wetting the particles with a fine mist of the liquidcomposition.

5) Drying the spherical particles in an oven under a stream of warm anddry air not exceeding 35° C. and 40% relative humidity.

6) Separating the microspheres according to the desired sizes using U.S.Standard sieve screens.

7) Coating the desired and classified microspheres (for example, in the16 to 20 mesh and separately in the 30 to 60 mesh size range) with anacid-resistant polymer in fluidized bed coating equipment, or in aconventional coating pan according to standard operating procedures asdescribed in the manufacturer's instruction manual.

8) Drying the polymer coated microspheres in an oven under a stream ofwarm and dry air not exceeding 35° C. and 40% relative humidity untilall the volatile substances (moisture and solvents) are removed.

The following examples will further serve to illustrate the compositionsof the present invention wherein the compositions and the process ofpreparing them will be described with reference to microsphere forms;however, it is to be noted that the microtablet form of the compositionand the process of making it is also intended to be covered by thepresent invention. The process of making the microtablet form of thecomposition is as follows:

a) blending dry, powdery ingredients selected from the group consistingof (i) from about 10 to about 90% w/w of a digestive enzyme from thegroup consisting of pancreatic proteases, lipases, nucleases andamylase; (ii) from about 0 to about 70% w/w of a salt of UDCA from thegroup consisting of sodium, potassium, ferrous, ammonium, tromethamine,ethanolamine, diethanolamine and triethanolamine; (iii) a bufferingagent selected from the group consisting of about 5 to about 40% sodiumcarbonate (anhydrous powder), sodium bicarbonate, potassium carbonate,ammonium carbonate, tromethamine, tris-carbonate(Di[tris(hydroxymethyl)amino-methane]carbonate), tris-glycine buffer(0.25 mole tris-base and 1.92 mole of glycine, pH 8.3), di-, tri-, andpoly-arginine in the molecular range of 350 to 50,000 daltons, di-, tri-and poly-lysine in the molecular range of 290 to 15,000 daltons,diethylamine and triethanolamine; (iv) from about 0.3 to about 15% w/wof an adhesive polymer selected from the group consisting ofhydroxypropyl cellulose, polyvinylpyrrolidone, cellulose acetate,phthalate, hydroxypropyl cellulose, and methyl cellulose; (v) of fromabout 0 to about 16% w/w a disintegrant selected from the groupconsisting of starch, modified starches, microcrystalline cellulose andpropylene glycol alginate;

b) wetting said blended ingredients with a liquid to cause the blend tostick together, wherein said liquid is selected from the groupconsisting of: 1%-25% w/w ethanol/75%-99% w/w 2-propanol/0.05%-1.5% w/wwater; 98%-99% w/w 2-propanol/0.05%-1.5% w/w water; and 1%-25% w/wmethanol/0.05%-1.5% w/w water/75%-98% w/w 2 propanol/1%-5% w/wethylacetate;

c) granulating or extruding the liquid-wetted blend through a 10 or 18mesh S/S screen;

d) drying the granulates or extruded particles;

e) admixing a lubricant, such as talc, stearic acid or magnesiumstearate in the amount of 0.1 to 2% based on the total weight of thecomposition with the granulated or extruded particles;

f) compressing the particles into microtablets of an average diametersize of from about 1.0 to about 2.5 mm;

g) coating the microtablets with a gastric acid-resistant polymer thatdisintegrates under neutral or slightly basic conditions; and

h) drying the polymer-coated microtablets.

EXAMPLE I Generalized Formula Composition (polymer coated)

    ______________________________________                                        Ingredients         % w/w                                                     ______________________________________                                        Disintegrant          0-16                                                    Salt of Bile acid     0-75                                                    Buffering agent     5.0-40                                                    Enzymes               0-90                                                    Adhesive Polymer    0.3-15                                                    Polymer coat/talc mixture                                                                         7.0-15                                                    ______________________________________                                    

EXAMPLE II Formula Composition

    ______________________________________                                                         IIA (uncoated)                                                                            IIB (coated)                                     Ingredients      % w/w       % w/w                                            ______________________________________                                        Disintegrant     3.0         2.7                                              Sodium Ursodeoxycholic acid                                                                    5.3         4.7                                              Buffering agent (anhydrous)                                                                    20.0        19.9                                             Enzymes          70.3        60.7                                             Adhesive Polymer 1.4         1.3                                              Polymer coat/talc mixture    10.7                                             ______________________________________                                    

EXAMPLE III Formula Composition

    ______________________________________                                                         IIIA (uncoated)                                                                           IIIB (coated)                                    Ingredients      % w/w       % w/w                                            ______________________________________                                        Disintegrant     3.0         2.6                                              Sodium-Ursodeoxycholic acid                                                                    70.0        60.9                                             Buffering agent (anhydrous)                                                                    10.0        8.7                                              Enzymes          10.3        9.0                                              Adhesive Polymer 6.7         5.8                                              Polymer coat/talc mixture    13.0                                             ______________________________________                                    

EXAMPLE IV Formula Composition

    ______________________________________                                                         IVA (uncoated)                                                                            IVB (coated)                                     Ingredients      % w/w       % w/w                                            ______________________________________                                        Disintegrant     2.0         1.8                                              Potassium-Ursodeoxycholic                                                                      1.5         1.4                                              acid                                                                          Buffering agent (anhydrous)                                                                    8.0         7.3                                              Enzymes          84.5        77.5                                             Adhesive Polymer 4.0         3.7                                              Polymer coat/talc mixture    8.3                                              ______________________________________                                    

EXAMPLE V Pancreatic Enzyme/Bile Salt Composition

    ______________________________________                                        Ingredients           % w/w                                                   ______________________________________                                        Bile salt starting seed (20-40 mesh)                                                                12.8                                                    Disintegrant          2.3                                                     Buffering agent (anhydrous)                                                                         9.1                                                     Enzymes               60.0                                                    Adhesive polymer mixture                                                                            5.1                                                     Polymer coat/talc mixture                                                                           10.7                                                    ______________________________________                                    

The microspheres of Example V were prepared by employing a conventionalcoating pan. The microspheres were built-up to larger particles sizes byplacing the buffered bile salt starting seeds in a rotating coating pan,wetting the microspheres with the liquid/adhesive polymer-containingmixture, followed by slowly dusting the buffered enzyme/disintegrantcomposition over the tumbling and flowing buffered-bile salt-containingseeds. The above steps were repeated until the seeds were built-up intomicrospheres having diameters in the range of 10 to 20 mesch, preferably14 to 16 mesh.

The bile salt starting seeds in Example V were prepared as outlined inExample VII. Suitable bile acids, bile salts and bile acid esters toprepare starting seeds in the particle size range of 20-60 mesh are:Ursodeoxycholic acid; sodium, potassium, ferrous and ammonium salts ofursodeoxycholic acid; ethyl and propyl esters of ursodeoxycholic acid;glycyl and tauroursodeoxycholic acid; sodium, potassium, ferrous andammonium salts of glycyl and tauroursodeoxycholate; N-methylglycylursodeoxycholate and N-methyltauroursodeoxycholate.

                  TABLE I                                                         ______________________________________                                        Distribution of the Microspheres According to Sizes                                             Example IIB  Example IIIB                                   Mesh Size                                                                             (mm)      Microspheres (%)                                                                           Microspheres (%)                               ______________________________________                                        10      2.00      --           3.5                                            20      0.84      10.0         57.0                                           40      0.42      53.8         32.7                                           60      0.25      28.6         5.2                                            80      0.177     7.6          1.6                                            ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Moisture Content & Stability of the Microspheres                                     Moisture   Stability Moisture Stability                                Mesh   Content (%)                                                                              (4 mo, %)*                                                                              Content (%)                                                                            (4 mo. %)*                               Size   IIB              IIIB                                                  ______________________________________                                        20     1.1        99        1.6      98                                       40     0.9        98        1.9      96                                       60     0.8        100       2.5      95                                       80     0.9        98        2.7      85                                       ______________________________________                                         *Lipase, Amylase and Protease Activities assayed according to USP XXII.  

Preparation of Salts of Bile Acids

In general, a bile acid, such as ursodeoxycholic acid, cholic acid,deoxycholic acid, chenodeoxycholic acid or their glycyl, taurine,methylglycyl and methyltaurine conjugates, was converted to the sodiumor potassium salt by dissolving the bile acid in a suitable solvent andtitrated with a water soluble alkaline hydroxide, carbonate orbicarbonate solution (e.g. sodium hydroxide, sodium carbonate, sodiumbicarbonate, potassium hydroxide, potassium carbonate, potassiumbicarbonate solutions) until the pH has reached pH 8.6. The solvent wasremoved by evaporation or by distillation and the bile-salt wasrecovered from the media by spray drying or by lyophilizing theremaining solution.

The preparation of the salts of bile acids was illustrated hereunder bythe preparation of the salts of ursodeoxycholic acid. It is to be noted,however, that the preparaton of the other salts of bile acids isanalogous to the preparation of the salts of ursodeoxycholic acid.

EXAMPLE VI

20 g of UDCA was dissolved in 100 ml of alcohol (methanol, ethanol,isopropanol or an other suitable alcohol that was easily removed afterUDCA has been neutralized) and a 10%-30% solution of hydroxide,bicarbonate or carbonate solution of Na or K was added to the reactionmixture, with rigorous mixing. The UDCA solution was titrated until thepH reached 8.6. The alcohol was removed from the reaction mixture on arotary evaporator, and the aqueous solution was processed to recover thesolid Na-UDCA by lyophilization or by spray-drying.

In another modification of preparing salts of UDCA an alcoholic solutionof UDCA is mixed with an alcoholic solution of sodium methoxide orsodium ethoxide, followed by evaporation of the alcoholic solvent andprecipitation of the Na-UDCA from the concentrated solution by addingthe Na-UDCA to ice-cold acetone in a ratio of 1:5 to 1:10. Collectingthe NaUDCA crystals by vacuum filtration, washing the crystals withice-cold acetone followed by air drying the crystals overnight. ExampleVI(a) illustrates this process.

EXAMPLE VI(a)

1000 g of ursodeoxycholic acid (UDCA) (2.55 mole) is dissolved in 2000ml of methanol. 500 ml of a methanolic solution of sodium methoxide(NaOCH₃) containing 138 g of sodium methoxide (2.55 mole) is then addedto the UDCA-containing solution. The so-obtained solution is stirred forone hour to assure complete neutralization. The alcohol is removed fromthe reaction mixture on a rotary evaporator and the syrupy liquidcontaining the NaUDCA (500 ml) is added to a rigorously mixed ice-coldacetone (2000 to 5000 ml) solution. The resulting white crystals ofNaUDCA are collected by vacuum filtration, washed with ice-cold acetone,followed by air drying the crystals in a tray drier overnight at 40° to80° C.

EXAMPLE VII Preparation of Bile Salt Starting Seeds

    ______________________________________                                                      % w/w                                                           ______________________________________                                        Bile Salt       50.0 to 70.0                                                  Disintegrant    10.0 to 16.0                                                  Buffering agent  8.0 to 18.0                                                  Adhesive polymer                                                                              12.0 to 16.0                                                  ______________________________________                                    

The process of making the bile salt-containing starting seeds consistedof: 1) blending the bile salt, disintegrant and the buffering agenttogether for 10-minutes; 2) spraying the composition with the adhesivepolymer mixture until the powdery blend agglomerated; and 3a) extrudingthe liquid moistened composition through a 10 or 18 mesh S/S screenusing an oscillating/reciprocating extruder or a twin-screw extruder;3b) build-up the granules into microspheres in a fluidized bed unit; or3c) a rotating pelletizing pan as described in Example V. The subsequentprocessing steps were the same as outlined in Steps (3) through (6) inthe "Detailed Description of the Invention".

EXAMPLE VIII Buffered Pancreatic Enzyme Formula Composition

    ______________________________________                                                          IVA (uncoated)                                                                            IVB (coated)                                    Ingredients       % w/w       % w/w                                           ______________________________________                                        Disintegrant      0           0                                               Potassium-Ursodeoxycholic acid                                                                  0           0                                               Buffering agent (anhydrous)                                                                     25          21.8                                            Enzymes           69          60                                              Adhesive Polymer  6           5.2                                             Polymer coat/talc mixture     13                                              ______________________________________                                    

Referring to ingredients used in the above examples:

Suitable Disintegrant in Examples I through V and Example VII are:ECPLOTAB (Mendell, Inc.), microcrystalline cellulose, and propyleneglycol alginate (Kelco Co.).

Suitable Buffering Agents in Examples I through V and Examples VII andVIIII are: sodium carbonate (anhydrous powder), sodium bicarbonate,potassium carbonate, and ammonium carbonate, tromethamine,diethanolamine, triethanolamine, tris-carbonate, tris-glycine buffer,di-, tri-, and poly-arginine, di-, tri-, and poly-lysine.

Suitable Enzymes in Examples I through V and Examples VII and VIII are:Pancreation, Pancrelipase and Pancreation concentrates of high potency.

Suitable Bile Salts in Examples I through V and Examples VII and VIIIare: sodium, potassium, ferrous and ammonium salts of ursodeoxycholate,glycylursodeoxycholate, taurinoursodeoxycholate,N-methylglycyldeoxycholate and N-methyltauroursodeoxycholate; organiccomplexes of tromethamine, diethanolamine and triethanolamine ofursodeoxycholate, glycylursodeoxycholate and taurinoursodeoxycholate;N-methylglycylursodeoxycholate; and N-methyltaurinoursodeoxycholate.

Suitable Adhesive Polymeric Agents in Example I through V and ExamplesVII and VIII are: Hydroxypropyl cellulose (KLUCEL HF, Hercules Co.),polyvinyl pyrrolidone (PLASDONE, GAF Co.), a 60:40 blend of methylcellulose and ethyl cellulose (Dow Chem. Co.), Hydroxypropyl methylcellulose (Grades 50 and 55, Eastman Kodak Co.), cellulose acetatephthalate (Eastman Kodak Co.) and propylene glycol alginate (Kelco Co.).

Suitable Acid-Resistant Polymers to coat the microspheres in Example Ithrough V and Examples VII and VIII are: Hydroxypropyl methyl cellulosephthalate, Grades 50 and 55 (Eastman Kodak Co., or Shin-Etsu ChemicalCo., Ltd.), AQUATERIC® aqueous enteric coating polymer dispersion (FMCCorp.), EUDRAGIT®) acrylic based polymeric dispersion (Rohm Pharma GMBH,Germany), and cellulose acetate phthalate (Eastman Kodak Co.).

Example IX will further illustrate the composition of the acid-resistantpolymer-coating:

EXAMPLE IX

    ______________________________________                                                                 % w/w                                                ______________________________________                                        Hydroxypropyl methyl cellulose phthalate*                                                                7.4                                                Diethyl phthalate or propylene glycol monostearate                                                       2.0                                                (MYVEROL P-06)                                                                2-Propanol                 45.2                                               Ethylacetate               45.2                                               Talc, USP                  0.2                                                ______________________________________                                         *When the hydroxypropyl methyl cellulose phthalate was replaced with          cellulose acetate phthalate an equally suitable acidresistant polymer         coating was obtained, as long as, talc was also included in the               composition. The presence of tale with the film forming polymer caused th     deposition of an acidimpermeable polymer coat. When AQUATERIC ® or        EUDRAGIT ® aqueous enteric coating polymer dispersion was employed in     place of cellulose acetate phthalate (CAP) or hydroxypropyl methyl            cellulose phthalate (HPMCP), the microspheres were first sealed with an       initial thin layer coating with CAP or HPMCP (2-4% w/w of the                 microspheres), followed by a secondary coating with an aqueous polymeric      latex dispersion (for example, AQUATERIC ® or EUDRAGIT ® ). The       employment of the aqueous coating composition as a secondary coating is       important to reduce the evaporation of solvents into the atmosphere and       thus reduce environmental pollution.                                     

The total amount of the composition required to be administered to abicarbonate/enzyme/bile salt deficient patient will vary with theseverity of the conditions, age and other physical characteristics ofthe patient. The physicians will prescribe the total amount, the dosageand the frequency of dosage administration on a patient by patientbasis. Generally, for enzyme/bile salt deficient patient from about 0.5to about 1.5 grams of the composition are administered with each majormeal, three times a day. Larger amounts may, however, be required forcertain conditions, such as for dissolving gallstones.

For ease of administration of the compositions it is preferred to usegelatin capsules containing about 0.2 to 0.5 grams microspheres ormicrotablets. Gelatin capsules which disintegrate in the acidicenvironment of the stomach are well-known and utilized in the prior art.Microtablets are of small size, having a diameter between about 1 to 5mm and a thickness between 0.5 to 4 mm. The microtablet is prepared byconventional tableting procedure. However, the compositions of thepresent invention in the form of very small particle sizes may be usedper se. The microspheres shown in Example IIB and IIIB (Table I) are inthe 10 to 80 mesh size range. Table I shows that 100% of themicrospheres of Example IIB were in the 20 mesh to 80 mesh size range(0.84 to 0.149 mm) and 89.7% of the coated particles IIIB were in therange of 20 to 40 mesh size range (0.84 to 0.42 mm). Young children oradults with certain diseases are unable to swallow big gelatin capsules.Microspheres of very small size of the present invention could then beadministered to the patients with liquid food, such as milk, and applesauce and with semi-solid foods.

The invention, having been fully described, it will be apparent to oneskilled in the art that changes and modifications can be made theretowithout departing from the spirit and scope thereof.

What is claimed is:
 1. A buffered digestive enzyme/bile salt compositionfor the treatment of digestive enzyme/bile salt deficiency of mammalscomprising, by weight per weight percentages based on the total weightof the composition:a) from about 10 to about 90.0% of a concentrate ofan enzyme selected from the group consisting of pancreatin, pancreaticproteases, pancreatic lipases, pancreatic nucleases and pancreaticamylases; b) from about 0.3 to about 75% of a bile salt in powder form;c) from 7.3 of a buffering agent selected from the group consisting ofanhydrous sodium carbonate, sodium bicarbonate, potassium carbonate,ammonium carbonate, tromethamine,di(tris(hydoxymethyl)aminomethane)carbonate, tris-glycine, di-argininein the molecular weight range of 350 to 50,000 Daltons, tri-arginine inthe molecular weight range of 350 to 50,000 Daltons, poly-arginine inthe molecular weight range of 350 to 50,000 Daltons, di-lysine in themolecular weight range of 290 to 15,000 Daltons, tri-lysine in themolecular weight range of 290 to 15,000 Daltons, poly-lysine in themolecular weight range of 290 to 15,000 Daltons, diethylamine andtriethanolamine; d) from about 0.9 to about 16% of a disintegrantselected from the group consisting of starch, modified starches,microcrystalline cellulose and propylene glycol alginate; e) from about0.3 to about 15.0% of an adhesive polymer selected from the groupconsisting of hydroxypropyl cellulose, polyvinylpyrrolidone, celluloseacetate phthalate, methyl cellulose and propylene glycol alginate; andf) from about 7.0 to about 15% of an non-porous, gastric acid-resistantand pharmaceutically acceptable polymer-coating which contains fromabout 0.2 to about 2% talc and which is insoluble in the pH range offrom about 1.5 to about 5 but is soluble in the pH range of from about5.5 to about
 9. 2. The composition of claim 1 wherein said concentrateof an enzyme is selected from the group consisting of pancreatin,pancrelipase, trypsin, chymotrypsin, chymotrypsin B, pancreatopeptidase.carboxypeptidase A, carboxypeptidase B, glycerol ester hydrolase,phospholipase A2, sterol ester hydrolase, ribonuclease,deoxyribonuclease, α-amylase, papain, chymopapain, bromelain, ficin,β-amylase, cellulase, and β-galactosidase.
 3. The composition of claim 1wherein the buffer component of said buffered digestive enzyme/bile saltis selected from the group consisting of anhydrous sodium carbonate,sodium bicarbonate, potassium carbonate, potassium bicarbonate, ammoniumcarbonate, tromethamine, tris-carbonate, tris-glycine, di-arginine,tri-arginine, poly-arginine, di-lysine, tri-lysine, poly-lysine,ethanolamine, diethanolamine and triethanolamine.
 4. The composition ofclaim 1 wherein said bile salt is selected from the group consisting ofsodium-ursodeoxycholate, sodium glycylursodeoxycholate,potassium-ursodeoxycholate, potassium glycylursodeoxycholate,ferrous-ursodeoxycholate, ferrous glycylursodeoxycholate,ammonium-ursodeoxycholate, ammonium glycylursodeoxycholate,sodium-tauroursodeoxycholate, sodium-N-methylglycylursodeoxycholate,potassium-tauroursodeoxycholate,potassium-N-methylglycylursodeoxycholate, ferrous-tauroursodeoxycholate,ferrous-N-methylglycylursodeoxycholate, ammonium-tauroursodeoxycholate,ammonium-N-methylglycylursodeoxycholate,sodium-N-methyltauroursodeoxycholate,potassium-N-methyltauroursodeoxycholate,ferrous-N-methyltauroursodeoxycholate,ammonium-N-methyltauroursodeoxycholate, sodium-cholate,sodium-deoxycholate, potassium-cholate, potassium-deoxycholate,ferrous-cholate, ferrous-deoxycholate, ammonium-cholate,ammonium-deoxycholate, sodium-chenodeoxycholate, sodium-glycylcholate,potassium-chenodeoxycholate, potassium-glycylcholate,ferrous-chenodeoxycholate, ferrous-glycylcholate,ammonium-chenodeoxycholate, ammonium-glycylcholate, sodium-taurocholate,sodium-N-methylglycylcholate, potassium-taurocholate,potassium-N-methylglycylcholate, ferrous-taurocholate,ferrous-N-methylglycylcholate, ammonium-taurocholate,ammonium-N-methylglycylcholate, sodium-N-methyltaurocholate,sodium-glycyldeoxycholate, potassium-N-methyltaurocholate,potassium-glycyldeoxycholate, ferrous-N-methyltaurocholate,ferrous-glycyldeoxycholate, ammonium-N-methyltaurocholate,ammonium-glycyldeoxycholate, sodium-taurodeoxycholate,sodium-N-methylglycyldeoxycholate, potassium-taurodeoxycholate,potassium-N-methylglycyldeoxycholate, ferrous-taurodeoxycholate,ferrous-N-methylglycyldeoxycholate, ammonium-taurodeoxycholate,ammonium-N-methylglycyldeoxycholate, sodium-N-methyltaurodeoxycholate,sodium-N-methylglycylchenodeoxycholate,potassium-N-methyltaurodeoxycholate,potassium-N-methylglycylchenodeoxycholate,ferrous-N-methyltaurodeoxycholate,ferrous-N-methylglycylchenodeoxycholate,ammonium-N-methyltaurodeoxycholate,ammonium-N-methylglycylchenodeoxycholate,sodium-N-methyltaurochenodeoxycholate,potassium-N-methyltaurochenodeoxycholate,ferrous-N-methyltaurochenodeoxycholate;ammonium-N-methyltaurochenodeoxycholate, ethyl esters ofursodeoxycholate, propyl esters of ursodeoxycholate,sodium-glycylchenodeoxycholate, potassium-glycylchenodeoxycholate,ferrous-glycylchenodeoxycholate, ammonium-glycylchenodeoxycholate,sodium-taurochenodeoxycholate, potassium-taurochenodeoxycholate,ferrous-taurochenodeoxycholate, ammonium-taurochenodeoxycholate.
 5. Amethod for treating digestive enzyme-bile salt deficiency in mammalscomprising orally administering an effective amount, sufficient toeffect a reduction in digestive enzyme/bile salt deficiency, of thecomposition of claim
 1. 6. The method of claim 5 wherein said treatmentis to effect a reduction in digestive enzyme/bile salt deficiency inpancreatic enzyme insufficiency, or impaired liver function, orinsufficient absorption of dietary iron and cholesterol, or cysticfibrosis, or presence of gallstones.
 7. The method of claim 5 whereinabout 0.5 to 1.5 gms of the composition is administered to a digestiveenzyme/bile salt deficient patient with each meal three times a day. 8.The method of claim 7 wherein said composition is administered in anacid soluble capsule containing from about 0.2 to about 0.5 grams ofmicrospheres or microtablets.
 9. The method of claim 7 wherein saidcomposition is administered admixed with a liquid or a semi-solid food.10. The method of claim 5 wherein said treatment is to effect areduction in digestive enzyme/bile salt deficiency in digestivedisorders.